Anticonvulsive pharmaceutical compositions

ABSTRACT

The present invention pertains to pharmaceutical compositions including, as the active ingredients, a combination of vigabatrin and of at least one glutamate receptor activating substance.

FIELD OF THE INVENTION

The present invention relates to anticonvulsive pharmaceuticalcompositions having reduced undesirable effects comprising theanticonvulsive agent vigabatrin in combination with at least oneglutamate receptor activating substance.

BACKGROUND OF THE INVENTION

Epilepsy is a general term describing a group of central nervous systemdisorders that are characterized by recurrent seizures that are theoutward manifestation of excessive and/or hyper-synchronous abnormalelectrical activity of neurons of the cerebral cortex and other regionsof the brain. This abnormal electrical activity can be manifested asmotor, convulsion, sensory, autonomic, or psychic symptoms.

Epilepsy is one of the more common neurological disorders and affectsmillions of people worldwide, and over 2.5 million individuals in theUnited States.

It is believed that the characteristic seizures of epilepsy are causedby the disordered, synchronous, and rhythmic firing of brain neurons.The neurons can fire at up to four times their normal rate. As a result,epileptic seizures are an overstimulation of the normal neuronalprocesses that control brain function.

Even though existing antiepileptic drugs can render 80% of newlydiagnosed patients seizure free, a significant number of patients havechronic intractable epilepsy causing disability with considerablesocioeconomic implications.

Anti-epileptic drugs are available for treating epilepsies, but theseagents have a number of shortcomings. For instance, the agents are oftenpoorly soluble in aqueous and biological fluids or are extremelyhygroscopic. Of even greater importance is that patients often becomerefractory to a drug over time. In addition, many anti-epileptic agentscause unwanted side effects, neurotoxicities, and drug interactions.Even while being treated with one or a combination of the anti-epilepticdrugs currently in clinical use, 30% of epileptic patients stillexperience seizures. As more anti-epileptic drugs are developed, theclinician will have expanded pharmaceutical options when designing aneffective treatment protocol for each patient.

Vigabatrin, which was developed as an inhibitor of gamma-aminobutyricacid transaminase, was one of the most promising novel anticonvulsantactive ingredients. However, vigabatrin was shown to induce highlysevere undesirable effects, such as an irreversible constriction of thevisual field. The constriction of the visual field induced by vigabatrinis asymptomatic when it is restricted to the nasal quadrant, until itextends to more central areas. Furthermore, visual defects induced byvigabatrin are not limited to the constriction of the visual field butalso includes dysfunction of central vision with a reduction of visualacuity, a loss of color discrimination and of contrast sensitivity. Anarrest of a therapeutical treatment with vigabatrin allows astabilization of the visual loss but very rarely induces any recovery.

However, because epileptic seizures are always very handicapping and maybe lethal, vigabatrin is still prescribed.

There is thus a need in the art for improved anticonvulsive, includinganti-epileptic, pharmaceutical compositions comprising vigabatrin, whichwould be endowed with reduced or no undesirable effects.

SUMMARY OF THE INVENTION

It is provided according to the present invention novel anticonvulsivepharmaceutical compositions comprising vigabatrin, which compositionspossess reduced undesirable effects, as compared with thevigabatrin-based pharmaceutical compositions known in the art.

One object of the present invention consists of a pharmaceuticalcomposition comprising, as the active ingredients, a combination ofvigabatrin and of at least one substance inducing an activation of aglutamate receptor within the retina tissue.

This invention also pertains to a method for treating convulsivedisorders, including epilepsy, comprising a step of administering, to apatient in need thereof, a combination of vigabatrin and of at least onesubstance inducing an activation of a glutamate receptor within theretinal tissue.

According to the present invention, substances inducing an activation ofglutamate receptor within the retina tissue encompass glutamate,glutamate derivatives, glutamate metabolites and glutamate analogues,inhibitors of glutamate reuptake, agonists of glutamate receptors andsubstances increasing glutamate release.

DETAILED DECRIPTION OF THE INVENTION

Surprisingly, it has been found according to the invention that theprogressive and irreversible loss of visual acuity of patients treatedwith vigabatrin is associated with a local decrease in glutamateconcentration within the retinal tissue, the said decrease in glutamateconcentration inducing retinal plasticity with formation of ectopicsynapses and induction of photoreceptor dysplasia.

More precisely, the inventors have shown that all ON bipolar cells(bipolar cells depolarizing at the light onset) and horizontal cellsundergo plasticity following treatment with vigabatrin at both rod andcone photoreceptor terminals. Rod photoreceptors were also found towithdraw their terminals toward their cell bodies. Photoreceptorwithdrawal and dendritic growth of postsynaptic neurons are associatedwith a loss of synaptic transmission between photoreceptors and theirpostsynaptic neurons. The plasticity of retinal neurons at thephotoreceptor synapses that is shown herein demonstrated a defect insynaptic transmission from photoreceptors to postsynaptic neurons duringthe vigabatrin treatment. The results obtained by the inventors are thusfully consistent with a reduction in glutamate and glutamineconcentration caused by vigabatrin because photoreceptors areglutamatergic neurons. This reduction of glutamate concentration maycontribute significantly to the formation of ectopic synapses, withadditional mechanisms that may contribute to photoreceptor dysplasia.These results are fully corroborated by the additional inventorsfindings showing that the vigabatrin-induced retinal plasticity isprevented by maintaining the vigabatrin-treated individuals in darkness,thus in conditions wherein a sufficient rate of glutamate release ispreserved although significantly lower than in normal conditions.Glutamate release is indeed maximum in dark-adapted conditions and it isreduced by light in a linear function of the light intensity.

The inventors findings that vigabatrin induces a local decrease in thelocal glutamate release within the retinal tissue, which decrease is—atleast partly—responsible from the deleterious side-effects of thisanticonvulsant substance, have allowed them to design improvedvigabatrin-based anticonvulsive methods and compositions with reducedside-effects, as compared to the methods and compositions includingvigabatrin that are already known in the art, wherein vigabatrin iscombined with at least one substance inducing an activation of glutamatereceptors within the retinal tissue, which substance may also be termedherein a “glutamate receptor activating substance”.

Vigabatrin consists of the International Common Name of4-amino-5-hexenoic acid, which may also be termed 4-aminohex-5-enoicacid (IUPAC designation), which chemical formula is C₆H₁₁NO₂, and whichhas the CAS Registry accession number 60643-86-9.

An object of the present invention consists of an anticonvulsivepharmaceutical composition comprising, as the active ingredients, acombination of (i) 4-amino-5-hexenoic acid and (ii) at least oneglutamate receptor activating substance.

By the expression “at least one substance”, it is intended herein “oneor more substance(s)”.

As used herein, an anticonvulsive pharmaceutical composition consists ofa pharmaceutical composition that is active for preventing and treatingconvulsive disorders. Convulsive disorders encompass epilepsy, tuberoussclerosis, as well as the convulsive disorders affecting patientsundergoing a drug addiction, including a drug addiction to heroin orcocaine.

As used herein, glutamate receptor activating substances encompass allsubstances that, when administered by local route or alternatively bysystemic route, cause an activation of glutamate receptors, whichinclude (i) substances inducing an increase in glutamate concentrationwithin the retina tissue, (ii) substances that inhibit reuptake ofsynaptically-released glutamate or that facilitate glutamatetransporter-mediated glutamate release, (iii) substances that directlyactivate glutamate receptors, e.g. agonists of the glutamate receptorsand (iv) substances that increase glutamate release by increasingglutamate uptake into synaptic vesicles or by facilitating synapticvesicular release of glutamate.

Substances inducing an increase in glutamate concentration encompassthose that cause an increase in the amount of glutamate that shouldnormally be present in the local inter-synaptic local areas included inthe retinal tissue, and more particularly in the local areas of theretinal tissue located between photoreceptor cells and postsynapticneurons.

Thus, glutamate receptor activating substances encompass (i) glutamate,substances that are transformed into glutamate by chemical or enzymaticreaction, substances comprising glutamate and substances generatingglutamate or glutamate-containing compounds after chemical or enzymereaction, (ii) substances that inhibit glutamate reuptake or favorglutamate transporter-mediated release by glutamate-releasing cells and(iii) substances that activate the glutamate receptor by mimicking orenhancing the glutamate activity, (iii) substances that facilitateglutamate vesicular release. In an anticonvulsive pharmaceuticalcomposition according to the invention, vigabatrin is combined to anyglutamate receptor activating substance(s). Notably, in ananticonvulsive pharmaceutical composition according to the invention,vigabatrin is combined with any one of those substances that are alreadyknown to exert a glutamate receptor activating activity.

In certain embodiments of an anticonvulsive pharmaceutical compositionaccording to the invention, the glutamate receptor activatingsubstance(s) is (are) selected from the group consisting of (i)glutamate, substances that are transformed into glutamate by chemical orenzymatic reaction, substances comprising glutamate and substancesgenerating glutamate or glutamate-containing compounds after chemical orenzymatic reactions, (ii) substances that inhibit glutamate reuptake byglutamate-releasing cells by inhibiting photoreceptor glutamatetransporters, by stimulating glutamate release through the transporters,(iii) substances that activate the glutamate receptor by mimicking orincreasing the glutamate activity and (iv) substances increasingglutamate vesicular release.

An anticonvulsive pharmaceutical composition according to the inventionmay comprise more than one glutamate receptor activating substance, soas to further reduce the deleterious effect of vigabatrin on the retinaltissue.

Thus, in certain embodiments of an anticonvulsive pharmaceuticalcomposition as described herein, the said composition may comprise,additionally to vigabatrin, 2, 3, 4, 5, 6, 7, 8, 9 or 10 distinctglutamate receptor activating substances, especially among thosebelonging to the various classes of glutamate receptor activatingsubstances that are listed above.

In certain embodiments, each of the more than one glutamate receptoractivating substance that is comprised in an anticonvulsivepharmaceutical composition according to the invention belongs to aspecific class of glutamate receptor activating substances, which classmay be distinct from the classes to which belong the at least one otherglutamate receptor activating substances combined therewith.

However, in most embodiments, an anticonvulsive pharmaceuticalcomposition according to the present invention comprises, in combinationto vigabatrin, 1, 2 or 3 distinct glutamate receptor activatingsubstances, wherein, optionally, each of the glutamate receptoractivating substance belongs to a specific class of glutamate receptoractivating substances among those listed above, which class is distinctfrom the class(es) to which belong the other(s) glutamate receptoractivating substance(s) combined therewith.

As it is already mentioned above, certain embodiments of ananticonvulsive pharmaceutical composition according to the inventioncomprise, as the glutamate receptor activating substance(s), a firstclass of substance(s) including glutamate, substances that aretransformed into glutamate by chemical or enzymatic reactions,substances comprising glutamate and substances generating glutamate orglutamate-containing compounds after chemical or enzymatic reactions.These glutamate receptor activating substances thus encompass glutamateper se, as well as glutamate derivatives, glutamate metabolites,glutamate-generating substances, glutamate analogues, including mixturesthereof.

This first class of glutamate receptor activating substances encompassesglutamate and glutamine, as well as salts thereof and mixtures thereof.

This first class of substances also encompasses alpha-ketoglutaric acid(AKG) as well as AKG-containing substances like ornitine-AKG,arginine-AKG, glutamine-AKG, glutamate-AKG and leucine-AKG, as well assalts of AKG, including salts of AKG with amino acids. This first classof substances also include salts of alpha-ketoglutaric acid (AKG)selected from the group consisting of mono-metal salt of AKG, di-metalsalt of AKG, mono-metal salt of glutamate and di-metal salt ofglutamate. These include for example calcium or natrium salts of AKG, aswell as calcium or natrium salts of glutamate.

This first class of substances further encompasses glutamate-containingdipeptides and glutamate-containing oligopeptides like, illustratively,L-alanyl-L-glutamate and L-glycyl-L-glutamate. These also includeglutamate polymers.

The second class of glutamate receptor activating substances comprisesinhibitors of glutamate reuptake or stimulators of glutamatetransporter-mediated release. The said inhibitors of glutamate reuptakeencompass any one of those inhibitors that are already known in the art,including inhibitors of glutamate transporter, like, illustratively,threo-3hydroxy-DL-aspartic acid (THA),(2S)-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), aminocaproic acid,and (2S,3S)-3-{3-[4-(Trifluoromethyl)benzoylamino]benzyloxy}aspartate orinhibitors of cystine-glutamate antiporters like(S)-4-carboxyphenylglycine.

The third class of glutamate receptor activating substances encompassesglutamate receptor agonists, i.e. substances that increase glutamatereceptor activity, especially in the retinal tissue. The glutamatereceptor agonists may be selected from the group consisting of indirect(allosteric) glutamate receptor agonists, direct glutamate receptoragonists, and, in some instances, partial glutamate receptor agonists.

As used herein, the term “glutamate receptor” encompasses variousglutamate receptors, including both ionotropic receptors (iGluR) andmetabotropic receptors (mGluR). lonotropic receptors intended hereininclude N-methyl-D-aspartic acid (NMDA) type, kainic acid (KA) type andalpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type,the two latter types of receptors being also currently termed asnon-NMDA-type glutamate receptors.

The third class of glutamate receptor activating substances, consistingof glutamate receptor agonists, include LY354740 which is aconformationally constrained analog of glutamate consisting of a mGluR⅔agonist, LY544344 which is a prodrug of LY354740, L-HCSA, L-homocysteinesulfinic acid; L-HCA, L-homocysteic acid L-CSA, L-cysteine sulfinicacid; L-CA, L-cysteic acid which are all agonist at all groups of mGluRreceptors, (S)-3,5-dihydroxyphenylglycine (DHPG), which is a group ImGluR receptor agonist, 2R,4R-APDC, which is a group II mGluR agonist,ACPT-1 and 2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid whichare two group III mGluR agonists(S)-2-(4-fluoro-phenyl)-1-(toluene-4-sulfonyl)-pyrrolidine (Ro67-7476);,diphenylacetyl-carbamicacid ethyl ester (Ro 01-6128) and(9H-xanthene9-carbonyl)-carbamic acid butyl ester (Ro 67-4853), whichare three mGluR1 positive allosteric modulators,3,3V-difluorobenzaldazine (DFB) and the second byN-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2Hisoindol-2-yl)methyl]phenyl}-2-hydroxybenzamide,which are two mGluR-5 positive allosteric modulators,N-(4-(2-methoxyphenoxy)phenyl)-N-(2,2,2-trifluoroethylsulfonyl)prid-3-ylmethylamine(LY487379), which is a mGluR2 positive allosteric modulator,(_)-N-Phenyl-7-(hydroximino)cyclopropa[b]chromen-1a-carboxamide((_)PHCCC), which is a positive allosteric modulator of mGluR4,(1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid and2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid which areagonists of mGluR4, L-(+)-2-Amino-4-phosphonobutyric acid and2-amino-4-(3-hydroxy-5-methylisoxazol-4-yl)butyric acid, which are twomGluR6 agonists, (R,S)3,4-DCPG, which is a mGluR8 receptor agonist,N-methyl-D-aspartic acid which is a NMDA receptor agonist,d-Cycloserine, which is a NMDA receptor agonist, glycine which is a NMDAreceptor agonist, spermidine which is a NMDA receptor agonist,milacemide which is a NMDA receptor agonist, cis-ACPD which is a NMDAreceptor agonist, homoquinolinic acid which is a NMDA receptor agonist,quisqualate which is a mGluR agonist, (S)-3,5-dihydroxyphenylglycine[(S)-DHPG which is a mGluR agonist,(_)-2-Oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate(LY379268) whichis a mGluR agonist, (2S,1′S,2′S)-2-(carboxycyclopropyl)glycine (L-CCG-I)which is an agonist of the metabotropic glutamate receptor,alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA),polyamines which are AMPA receptor agonists, S-(−)-5-fluorowillardinewhich is an AMPA receptor agonist, (RS)-Willardine which is an AMPAreceptor agonist, Ampakines which are AMPA receptor agonists, kainicacid (KA), domic acid which is a KA receptor agonist and SYM 2081 whichis a KA receptor agonist.

Another non-NMDA type glutamate receptor agonist dysiherbaine may alsobe used as a third class glutamate receptor activating substance, thesaid substance being disclosed in the U.S. Pat. No. 6,147,230.

Also, other NMDA receptor agonists such as(2S,1′R,2′R,3′R)-2-(2,3-dicarboxycyclopropyl)-glycine (DCG-I) and(2S,1′S,2′S,3′S)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-II) may alsobe used as a third class glutamate receptor activating substances, thesaid substances being disclosed in the U.S. Pat. No. 5,334,757, as wellas (2S,3R,4S)-carboxycyclopropylglycine as disclosed in the U.S. Pat.No. 4,959,493.

The fourth class of glutamate receptor activating substances encompassesstimulators of synaptic release, i.e. substances that increase glutamaterelease, especially in photoreceptors. The said stimulators of glutamaterelease encompass any one of those stimulators that are already known inthe art. The said stimulators of glutamate release may be selected from(a) agonists or antagonists of presynaptic receptors regulating calciumchannels, (b) compounds inducing vesicular release of glutamate and (c)molecules inducing a depolarization of photoreceptors (i.e.photoreceptor depolarizing compounds).

The fourth class of glutamate receptor activating substances, consistingof stimulators of glutamate release, includes(R,S)-alpha-methylserine-O-phosphate (MSOP), an antagonist of group IIImGluR receptors, theophylline (aminophylline), which is an Adenosinereceptor antagonists (S)PHPNECA, LUF5835 and LUF5845, which areAdenosine receptor A2 antagonists, Rp-cAMPS, which is an inhibitor ofprotein kinase A (PKA), Sildenafil and vardenafil, two inhibitors of thephodiesterase 6 (PDE6).

In certain embodiments of an anticonvulsive pharmaceutical compositionaccording to the invention, the said pharmaceutical composition furthercomprises at least one substance having an anti-ischemic effect. Any oneof the substances that are already known to exert an effect againstischemia, and especially an effect against retinal ischemia, areencompassed herein.

As used herein, “retinal ischemia” encompasses generally any disorderinvolving an hypoxic condition of the retinal tissue, especially anydisorder that reduces availability of blood, oxygen or other nutrientsto the retinal tissue and which can result in retinal tissuedisorganization and retinal cell death. Thus, retinal ischemiaencompasses hypoxic conditions of the retinal tissue, notably those thatare caused by a reduction of the arterial blood flow or the venous bloodflow to, or in, the retina, as well as those resulting from adysfunction of the retinal pigment epithelium in transferring glucoseand oxygen to photoreceptors.

In those embodiments of an anticonvulsive pharmaceutical compositionaccording to the invention, the said substance having an anti-ischemiceffect is preferably selected from the group consisting of anantioxidant substance, a free radical scavenger substance, a statin, an

ACE inhibitor, an AT-1 antagonist, a calcium channel inhibitor, a sodiumchannel blocker agent, a potassium channel activator agent, abeta-adrenergic blocking agent, an anesthetics substance, ananticonvulsive agent, a hormone, a vasodilatator agent, an α-receptorantagonist, a xanthine oxidase inhibitor, a cyclooxygenase inhibitor, aprotease inhibitor, an immunosuppressant agent and a mitochondrial ATPsensitive potassium opener agent.

Thus, in certain embodiments of an anticonvulsive pharmaceuticalcomposition that comprise, in addition to vigabatrin and one or moreglutamate receptor activating substance(s), also at least oneanti-ischemic substance, and illustratively 2, 3, 4, 5, 6, 7, 8, 9 or 10distinct anti-ischemic substances, especially among those belonging tothe various classes of anti-ischemic substances that are listed above.

In some of these embodiments, each of the more than one anti-ischemicsubstance that is comprised in an anticonvulsive pharmaceuticalcomposition according to the invention belongs to a specific class ofanti-ischemic substances, which class is distinct from the classes towhich belong the at least one other anti-ischemic substance combinedtherewith.

However, in most of these specific embodiments of an anticonvulsivepharmaceutical composition according to the present invention, the saidcomposition comprises, in combination to vigabatrin and the at least oneglutamate receptor activating substance, also 1, 2 or 3 distinctanti-ischemic substances, wherein, optionally, each of the anti-ischemicsubstance belongs to a specific class of anti-ischemic substances amongthose listed above, which class is distinct from the class(es) to whichbelong the other(s) anti-ischemic substance(s) combined therewith.

Illustratively, anti-ischemic substances may consist of antioxidantsubstances, such as antioxidant substances selected from the groupconsisting of glutathion, N-acetylcysteine, alpha-lipoic acid,resveratrol

(CAS registry no 501-36-0), ramelteon((S)-N-[2-(1,6,7,8-tetrahydro-2H-indeno-[5,4-b]furan-8-yl)ethyl]propionamide; CAS Registry no 196597-26-9), a retinoidcompound, an antioxidant vitamin, co-enzyme Q-10, beta carotene, uricacid, L-2-oxothiazolidine-4-carboxylic acid and melatonin.

The antioxidant vitamin may be selected from the group consisting ofα-tocopherol, vitamin B, vitamin C, vitamin D, vitamin E, vitamin K, andsalts thereof.

Other antioxidant substances may consist of antioxidant compoundsselected from the group consisting of a flavonoid, a polyphenol, aphytooestrogen or an extract from Ginkgo biloba.

Further antioxidant substances may consist of antioxidant compoundsselected from the group consisting of a SOD-like substance and acatalase-like substance.

Still further antioxidant substances may consist of free radicalscavenger substances selected from the group consisting of tirilazad,ebselen, ederavone and melatonin.

Pharmaceutical Compositions and Methods

Generally, a pharmaceutical composition according to the inventioncomprises the combination of vigabatrin with at least one glutamatereceptor activating substance and further also one or morephysiologically acceptable excipients.

In specific embodiments of pharmaceutical compositions according to theinvention, the said compositions further comprise one or moreanti-ischemic substances, as it is described above.

Vigabatrin, the at least one glutamate receptor activating substance,and optionally the at least one anti-ischemic substance, are comprisedin an anticonvulsive pharmaceutical composition in a “therapeuticallyeffective amount”, that is in an amount sufficient for the combinationof active ingredients to exert the expected anticonvulsive effect whileinducing no deleterious side effect, or side effects which are reducedas compared with the deleterious side effects induced by pharmaceuticalcompositions comprising vigabatrin without any glutamate receptoractivating substance nor any anti-ischemic substance.

Generally speaking, a “therapeutically effective amount”, or “effectiveamount”, or “therapeutically effective”, as used herein, refers to thatamount which provides a therapeutic effect for a given condition andadministration regimen. This is a predetermined quantity of activematerial calculated to produce a desired therapeutic effect inassociation with the required additive and diluent; i.e., a carrier, oradministration vehicle. Further, it is intended to mean an amountsufficient to reduce and most preferably prevent, a clinicallysignificant deficit in the activity, function and response of the host.Alternatively, a therapeutically effective amount is sufficient to causean improvement in a clinically significant condition in a host. As isappreciated by those skilled in the art, the amount of a compound mayvary depending on its specific activity. Suitable dosage amounts maycontain a predetermined quantity of active composition calculated toproduce the desired therapeutic effect in association with the requireddiluent; i.e., carrier, or additive.

The present invention also concerns methods for preventing or treatingconvulsive disorders comprising a step of administering (i) acombination of vigabatrin and of at least one glutamate receptoractivating substance or (ii) a pharmaceutical composition as definedabove, to an individual in need thereof.

The individuals in need of such treatments encompass those, either adultor child patients, which are susceptible to convulsive disorders,especially epilepsy.

Thus, another object of the present invention consists of a method forpreventing or treating convulsive disorders of a patient comprising astep of administering to a patient in need thereof a combination of (i)4-amino-5-hexenoic acid and (ii) at least one glutamate receptoractivating substance.

In some embodiments, the method above comprises a step of administeringto a patient in need thereof a combination of (i) 4-amino-5-hexenoicacid, (ii) at least one glutamate receptor activating substance and(iii) at least one anti-ischemic substance.

In certain embodiments, the said method comprises a step ofadministering to a patient in need thereof a pharmaceutical compositionthat is described in the present specification.

By “physiologically acceptable excipient or carrier” is meant solid orliquid filler, diluent or substance which may be safely used in systemicor topical administration. Depending on the particular route ofadministration, a variety of pharmaceutically acceptable carriers wellknown in the art include solid or liquid fillers, diluents, hydrotropes,surface active agents, and encapsulating substances.

Pharmaceutically acceptable carriers for systemic administration thatmay be incorporated in the composition of the invention include sugar,starches, cellulose, vegetable oils, buffers, polyols and alginic acid.Specific pharmaceutically acceptable carriers are described in thefollowing documents, all incorporated herein by reference: U.S. Pat. No.4,401,663, Buckwalter et al. issued Aug. 30, 1983; European PatentApplication No. 089710, LaHann et al. published Sep. 28, 1983; andEuropean Patent Application No. 0068592, Buckwalter et al. publishedJan. 5, 1983. Preferred carriers for parenteral administration includepropylene glycol, pyrrolidone, ethyl oleate, aqueous ethanol, andcombinations thereof.

Representative carriers include acacia, agar, alginates,hydroxyalkylcellulose, hydroxypropyl methylcellulose,carboxymethylcellulose, carboxymethylcellulose sodium, carrageenan,powdered cellulose, guar gum, cholesterol, gelatin, gum agar, gumarabic, gum karaya, gum ghatti, locust bean gum, octoxynol 9, oleylalcohol, pectin, poly(acrylic acid) and its homologs, polyethyleneglycol, polyvinyl alcohol, polyacrylamide, sodium lauryl sulfate,poly(ethylene oxide), polyvinylpyrrolidone, glycol monostearate,propylene glycol monostearate, xanthan gum, tragacanth, sorbitan esters,stearyl alcohol, starch and its modifications. Suitable ranges vary fromabout 0.5% to about 1%.

For formulating a pharmaceutical composition according to the invention,the one skilled in the art will advantageously refer to the last editionof the European pharmacopoeia or of the United States pharmacopoeia.

Preferably, the one skilled in the art will refer to the fifth edition“2005” of the European Pharmacopoeia, or also to the edition USP 28-NF23of the United States Pharmacopoeia.

The weight amount of the combination of active ingredients that iscontained in each dose of the pharmaceutical composition of theinvention will depend on the molecular weight of said therapeuticallyactive compound as well as on the weight amount that is effective ininhibiting or blocking the convulsive disorder. Effective amounts ofvigabatrin that are needed for preventing or treating convulsivedisorders are well known from the one skilled in the art.

For determining the appropriate amount of the glutamate receptoractivating substance(s) to be combined with vigabatrin, in a dose of apharmaceutical composition of the invention, the one skilled in the artmay advantageously refer to the effective amounts that are already knownor determined in the art for the anti-ischemic substance(s) that is(are) comprised therein.

Also, in certain embodiments, the appropriate amount of theanti-ischemic substance(s) to be combined with vigabatrin and theglutamate receptor activating substance(s), in a dose of apharmaceutical composition of the invention, the one skilled in the artmay advantageously refer to the effective amounts that are already knownor determined in the art for the anti-ischemic substance(s) that is(are) comprised therein.

The present invention is further illustrated by the examples below.

EXAMPLES A. Materials and Methods of the Examples

A.1. Animal treatment

BALB/c mice (16 treated, 10 controls) were purchased from Janvier (LeGenest-St-lsle, France) at six weeks. VGB dissolved in 0.9%

NaCl at 100 mg/m1 was intraperitoneally injected daily for 30 days at afinal concentration of 250 mg/kg as described for animal treatment ofepilepsy (Andre V, Ferrandon A, Marescaux C, Nehlig A. Vigabatrinprotects against hippocampal damage but is not antiepileptogenic in thelithium-pilocarpine model of temporal lobe epilepsy. Epilepsy Res.2001;47:99-117). This dose was slightly higher than that prescribed toadult patient (1-6 mg/kg), children (100 mg/kg), or infants (250 mg/kg)(Bialer M, Johannessen S I, Kupferberg H J et al. Progress report on newantiepileptic drugs: a summary of the Fifth Eilat Conference (EILAT V).Epilepsy Res. 2001;43:11-58).

A.2. Histology

Eye cups were fixed overnight at 4° C. in 4% (wt/vol) paraformaldehydein phosphate buffered saline (PBS) 0.01 M, pH 7.4. After cryoprotectionin PBS containing successively 10%, 20% and 30% sucrose at 4° C., thetissue was embedded in OCT (Labonord, Villeneuve d'Ascq, France).Vertical sections (8-10 μm thickness) were permeabilized for 5 minutesin PBS containing 0.1% Triton X-100 (Sigma, St. Louis, Mo.), rinsed, andincubated in PBS containing 1% bovine serum albumin (Eurobio, Les-Ulis,France), 0.1% Tween 20 (Sigma), and 0.1% sodium azide (Merck,Fontenay-Sous-Bois, France) for 2 hours at room temperature. The primaryantibody added to the solution was incubated for 2 hours at roomtemperature. The polycolonal antibodies were directed against theprotein kinase C alpha (PKCα) (1:2000, Sigma), VGluT1 (1:2000,Chemicon), Goα (K-20; 1:100, Santa Cruz), calbindin D-28K (1:500,Chemicon), mouse cone arrestin/ Luminaire junior (LUMIj) (1:20,000, ²⁷).The monoclonal antibodies were directed against Goα (1:2000, Chemicon),bassoon (1:100, StressGen) and calbindin D-28K (1:500, Sigma). Afterrinses, sections were incubated with the secondary antibody, goatanti-rabbit IgG or rabbit anti-mouse IgG conjugated to either AlexaTM594 or Alexa TM488 (1:500, Molecular Probes, Eugene, Oreg.) for 2hours. The dye, diamidiphenyl-indole (DAPI) was added to the lastincubated solutions. Sections were rinsed, mounted with Fluorsavereagent (Calbiochem, San Diege, Calif.) and viewed with a Leicamicroscope (LEICA DM 5000B) equipped with a Ropper scientific camera(Photometrics cool SNAP TM FX).

B. Results of the Examples Example 1 Retinal Changes inVigabatrin-Treated Mice

In rats, the major retinal changes consisted in a retinal architecturedisorganization, a major glial reaction, cone damages and photoreceptorapoptosis. Initially, we examined these previously observed retinalchanges in VGB-treated mice. Areas with a highly disorganized outernuclear layer (ONL) were also observed in VGB-treated albino mice butnot in all animals (5 of 16 treated mice). The results show thatphotoreceptor layer can become disorganized in VGB-treated mice, as wellas in VGB-treated rats.

To examine cone photoreceptors, these cells were immunolabelled with thespecific mouse cone arrestin antibody (Zhu X, Li A, Brown B et al. Mousecone arrestin expression pattern: light induced translocation in conephotoreceptors. Mol Vis. 2002;8:462-471). The results show the intenselabelling of cone outer segments and of their terminals with the conearrestin antibody. In VGB-treated mice, cone outer segments haddisappeared, whereas their terminals remained intensely stained and somecell bodies became positive. These observations indicated the presenceof cone damage in VGB-treated mice as in treated rats.

When the retinal sections of control albino mice were immunolabelledagaint GFAP, glial Muller cells were already intensely stained and thisGFAP staining was not increased by the VGB-treatment. These observationsindicated that retinal damages had similarities with those described inVGB-treated rats but with a reduced level of retinal gliosis.

To examine the reaction of inner retinal, ON bipolar cells were stainedwith the antibody directed against the protein Goα. In control animals,ON bipolar cells exhibited cell bodies with very short dendritesextending to the outer plexiform layer (OPL). By contrast, inVGB-treated animals, ON bipolar cells extended their dendrites deep intothe ONL.

To determine if both rod and cone ON bipolar cells were growingdendrites into the ONL, retinal sections were double immunolabelled withthe antibody directed against the protein kinase Cα (PKCα), whichidentify selectively ON rod bipolar cells. The results show that rod ONbipolar cells exhibited dendrites extending into the ONL. When PKCα andGoα double immunolabelled retinal sections were carefully examined, someGoα-positive dendrites that extended to the outer nuclear layer werefound to be PKCα negative indicating thereby that cone ON bipolar cellsalso extended their dendrites into the ONL. These observationsdemonstrated that both rod and cone ON bipolar cells underwentplasticity following the VGB treatment.

To investigate whether horizontal cells follow the same pattern ofplasticity, these were stained with an antibody directed againstcalbindin. In control retina, the labelling showed horizontal cells withtheir dendrites ramifying into the OPL as well as some amacrine cellbodies. In VGB-treated animals, horizontal cells extended theirdendrites deep into the ONL like ON bipolar cells. Results obtained withDAPI-stained photoreceptor nuclei showed that this neuronal plasticitywas observed in areas with no disorganization of the ONL. In fact,growing dendrites of postsynaptic neurons were present along thecomplete retinal sections, suggesting that these features of plasticityoccurred in postsynaptic neurons prior to the ONL disorganization.

Because bipolar cells and horizontal cells normally have their dendritictips associated at photoreceptor invaginating synapses, doubleimmunolabelling examined if they remain associated during theirdendritic sprouting. Although Goα-positive bipolar cell dendrites wereoften associated with calbindin-positive horizontal cell dendrites, afew Goα-positive dendrites appeared isolated from calbindin-positiveprocesses. These observations indicated that the dendritic growth inhorizontal cells and ON bipolar cells could occur independently in VGBtreated animals.

To determine whether the sprouting of postsynaptic neurons is related tochanges in photoreceptor synaptic terminals, we used antibodies againstbassoon, a protein of the photoreceptor synaptic ribbon, and againstVGLUT1, a protein of photoreceptor synaptic vesicles. On retinalsections of control animals, bassoon immunolabelling showed a punctuatedistribution restricted to the OPL.

In contrast, in VGB-treated animals, the punctuate labelling wasobserved to penetrate deep into the ONL. Similarly, the VGLUT1immunolabeling was restricted to the OPL in control animals whereas itwas observed deep into the ONL in VGB-treated animals. These resultsindicated that photoreceptor terminals were withdrawn into the ONL closeto photoreceptor nuclei. In contrast, cone photoreceptor terminalsappeared to remain in the OPL even in highly disorganized areas.

To confirm the absence of cone photoreceptor terminal withdrawal inareas with dendritic growth of postsynaptic neurons, retinal sectionswere immunolabelled with the cone arrestin antibody while ON bipolarcells were identified with the Goα-antibody. Although the OPL structurein VGB-treated mice was not as linear as in control animals, coneterminals remained in the OPL whereas Goα-positive dendrites of ONbipolar cells were already detected very deep into the ONL. Theseobservations suggested that rod but not cone photoreceptor terminalswere withdrawn toward their cell bodies.

To investigate whether withdrawing rod photoreceptor terminals keepcontact with their postsynaptic neurons, photoreceptor synapticterminals were immunolabelled with the VGLUT1 antibody while ON bipolarcells were identified with the Goα antibody. This double immunostainingshowed that although rod photoreceptor terminals were often facing asprouting bipolar cell dendrite, few VGLUT1-positive photoreceptorterminals were not associated to a growing bipolar cell dendrite. Tofurther examine whether rod photoreceptor synaptic ribbons had retractedaway from their postsynaptic neurons, photoreceptor synaptic ribbonswere immunolabelled with the bassoon antibody while rod ON bipolar cellswere stained with the PKCα antibody. Again, although mostbassoon-positive synaptic ribbons were facing a sprouting rod ON bipolarcell dendrite, a few synaptic ribbons were not contacted by a rodbipolar cell dendrite. These observations indicated that photoreceptorwithdrawal and dendritic growth of postsynaptic neurons were associatedwith a loss of synaptic transmission between photoreceptors and theirpostsynaptic neurons.

Example 2 Light Dependence of the Vigabatrin-Elicited Retinal Toxicity

Albino rats only are sensitive to the VGB-elicited retinal toxicity(Butler W H, Ford G P, Newberne J W. A study of the effects ofvigabatrin on the central nervous system and retina of Sprague Dawleyand Lister-Hooded rats. Toxicol Pathol. 1987;15:143-148). Therefore, wetested whether VGB retinal damages were due to light because the retinaof albino animals receives more than a log unit higher luminance higherthan that of pigmented animals (Lyubarsky A L, Daniele L L, Pugh E N,Jr. From candelas to photoisomerizations in the mouse eye by rhodopsinbleaching in situ and the light-rearing dependence of the majorcomponents of the mouse ERG. Vision Res. 2004;44:3235-3251). WhenVBG-treated animals were maintained in a dark room throughout thetreatment, bipolar cells were found not to grow dendrites in the ONL asin control animals. Furthermore, photoreceptors did not retract theirterminals in the ONL. Other retinal damages like ONL disorganizationwere not observed in these VGB-treated animals maintained in darknesswhereas animals under the 12h light/dark cycle showed the bipolar celldendritic growth and the photoreceptor terminal withdrawal. Theseresults indicated that maintaining the animals in darkness during VGBtreatment can prevent the retinal toxicity.

For summarizing the results of the Examples herein, it has been shownthat vigabatrin induced plasticity of postsynaptic neurons which isconsistent with a vigabatrin-induced defect in photoreceptor synaptictransmission. In VGB-treated animals, plasticity in neurons postsynapticto photoreceptors therefore suggests that photoreceptor synaptictransmission was impaired by the treatment. This VGB-inducedphotoreceptor impairment is further indicated by the rod terminalwithdrawal as observed in both retinal detachment and in bassoonknockout mice, in which ectopic synapses are also forming in the ONL.Considering cones, the photoreceptor impairment is supported by the lossof their outer segments although their terminals did not appear towithdraw. Therefore, the plasticity of retinal neurons at thephotoreceptor synapses demonstrates a defect in synaptic transmissionfrom photoreceptors, the photoreceptors consisting of glutamatergicneurons. More precisely, photoreceptors are glutamatergic neurons andcontinuously release glutamate in the dark with a modulation of thisrelease by light stimulation such that the glutamate concentration inthe synaptic cleft is a linear function of light intensity. The resultsshown in the Examples herein strongly suggest that the observed defectin photoreceptor synaptic transmission is related to a retinal reductionin glutamate and glutamine concentrations in vigabatrin-treated animals.This decrease in glutamate in vigabatrin-treated animals helpsexplaining the formation of ectopic synapses in the ONL. This decreasein glutamate concentration could therefore contribute significantly tothe formation of ectopic synapses, but with additional mechanisms thatmay contribute to photoreceptor dysplasia. The prevention of thisretinal plasticity when maintaining the animal in darkness is consistentwith this importance of the decrease in glutamate. Indeed, the rate ofglutamate release is maximum in darkness under normal conditions.Therefore, during the VGB treatment, if retinal plasticity results froman insufficient glutamate release at photoreceptor terminals,maintaining animals in darkness may preserve a sufficient rate ofglutamate release although significantly lower than in normalconditions. These observations induce that VGB retinal toxicity could besuppressed by locally increasing the glutamate concentration in theretina.

1. An anticonvulsive pharmaceutical composition comprising, as theactive ingredients, a combination of (i) 4-amino-5-hexenoic acid and(ii) at least one glutamate receptor activating substance.
 2. Thepharmaceutical composition according to claim 1, wherein the at leastone glutamate receptor activating substance is selected from the groupconsisting of glutamate, glutamate derivatives, glutamate metabolitesand glutamate analogues.
 3. The pharmaceutical composition according toclaim 2, wherein the at least one glutamate receptor activatingsubstance is selected from the group consisting of alpha-ketoglutaricacid (AKG), ornitine-AKG, arginie-AKG, glutamine-AKG, glutamate-AKG,leucine-AKG and salts of AKG with amino acids.
 4. The pharmaceuticalcomposition according to claim 2, wherein the at least one glutamatereceptor activating substance consists of a salt of alpha-ketoglutaricacid (AKG) selected from the group consisting of mono-metal salt of AKG,di-metal salt of AKG, mono-metal salt of glutamate and di-metal salt ofglutamate.
 5. The pharmaceutical composition according to claim 2,wherein the at least one glutamate receptor activating substance isselected from the group consisting of glutamate dipeptides and glutamateoligopeptides.
 6. The pharmaceutical composition according to claim 1,wherein the at least one glutamate receptor activating substanceconsists of an inhibitor of glutamate reuptake or that facilitateglutamate transporter-mediated glutamate release.
 7. The pharmaceuticalcomposition according to claim 1, wherein the at least one glutamatereceptor activating substance consists of a glutamate receptor agonist.8. The pharmaceutical composition according to claim 1, wherein the atleast one glutamate receptor activating substance consists of asubstance increasing the synaptic vesicular release of glutamate.
 9. Thepharmaceutical composition according to claim 1 which further comprisesat least one substance having an anti-ischemic effect.
 10. A method forpreventing or treating convulsive disorders of a patient comprising astep of administering to a patient in need thereof a compositioncomprising a combination of (i) 4-amino-5-hexenoic acid and (ii) atleast one glutamate receptor activating substance.
 11. A method forpreventing or treating convulsive disorders of a patient comprising astep of administering to a patient in need thereof a compositioncomprising a combination of (i) 4-amino-5-hexenoic acid and (ii) atleast one glutamate receptor activating substance, further comprising astep of administering to a patient in need thereof a pharmaceuticalcomposition according to claim
 2. 12. The method according to claim 10,wherein the said convulsive disorder consists of epilepsy.
 13. A methodfor preventing or treating convulsive disorders of a patient comprisinga step of administering to a patient in need thereof a compositioncomprising a combination of (i) 4-amino-5-hexenoic acid and (ii) atleast one glutamate receptor activating substance, further comprising astep of administering to a patient in need thereof a pharmaceuticalcomposition according to claim
 3. 14. A method for preventing ortreating convulsive disorders of a patient comprising a step ofadministering to a patient in need thereof a composition comprising acombination of (i) 4-amino-5-hexenoic acid and (ii) at least oneglutamate receptor activating substance, further comprising a step ofadministering to a patient in need thereof a pharmaceutical compositionaccording to claim
 4. 15. A method for preventing or treating convulsivedisorders of a patient comprising a step of administering to a patientin need thereof a composition comprising a combination of (i)4-amino-5-hexenoic acid and (ii) at least one glutamate receptoractivating substance, further comprising a step of administering to apatient in need thereof a pharmaceutical composition according to claim5.
 16. A method for preventing or treating convulsive disorders of apatient comprising a step of administering to a patient in need thereofa composition comprising a combination of (i) 4-amino-5-hexenoic acidand (ii) at least one glutamate receptor activating substance, furthercomprising a step of administering to a patient in need thereof apharmaceutical composition according to claim
 6. 17. A method forpreventing or treating convulsive disorders of a patient comprising astep of administering to a patient in need thereof a compositioncomprising a combination of (i) 4-amino-5-hexenoic acid and (ii) atleast one glutamate receptor activating substance, further comprising astep of administering to a patient in need thereof a pharmaceuticalcomposition according to claim
 7. 18. A method for preventing ortreating convulsive disorders of a patient comprising a step ofadministering to a patient in need thereof a composition comprising acombination of (i) 4-amino-5-hexenoic acid and (ii) at least oneglutamate receptor activating substance, further comprising a step ofadministering to a patient in need thereof a pharmaceutical compositionaccording to claim
 8. 19. A method for preventing or treating convulsivedisorders of a patient comprising a step of administering to a patientin need thereof a composition comprising a combination of (i)4-amino-5-hexenoic acid and (ii) at least one glutamate receptoractivating substance, further comprising a step of administering to apatient in need thereof a pharmaceutical composition according to claim9.